This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Spermatozoa that leave the mammalian testis are not motile and do not have the capability to fertilize an oocyte. Sperm maturation begins in the epididymis and continues in the female reproductive tract with the process of capacitation and finally the acrosome reaction. This maturation process is unique in that sperm are transcriptionally and translationally quiescent. Sperm maturation occurs in a "closed system" where changes involve primarily modification of existing proteins. One of the biggest changes that occur is the phosphorylation states of the many sperm proteins, and these changes appear to be required for sperm function and fertility. Consequently, we propose to investigate the system wide changes which occur to the phosphoproteome of the sperm. This will be approached two ways: 1) Using the proteomic data from epididymal sperm that we have already acquired from PNNL and informatics techniques to identify which proteins have specific phosphorylation motifs that can be phosphorylated and which kinases and phosphatases are present in the sperm proteome to act on these motifs, and 2) isolate sperm at various stages of maturation and subject them to phosphoproteomics analysis to identify important maturation-related phosphorylation events. These data will be used to assemble a system analysis of the sperm phospho-proteome. These experiments will fill a significant gap in our understanding of the phosphorylation network of sperm maturation. Given the importance of kinases and phosphatases as drug targets, this work promises rapid identification of potential non-hormonal contraceptive targets in the male.